Transport and Fixation of Inorganic Carbon during Photosynthesis in Cells of Anabaena Grown under Ordinary Air: III. Some Characteristics of the HCO3--Transport System in Cells Grown under Ordinary Air

In cells of cyanobacterium Anabaena variabilis grown under ordinaryair (low-CO₂ cells), the transport of both CO₂ and HCO₃^–was significantly enhanced by Na⁺. This effect was pronouncedas the external pH increased. When low-CO₂ cells were treatedwith an inhibitor of carbonic anhydrase (CA), only CO₂ transportbut not HCO₃^– transport, was inhibited. The initial rateof photosynthetic carbon fixation as a function of the concentrationof internal inorganic carbon (IC) was practically the same irrespectiveof whether CO₂ or HCO₃^– was externally supplied. Theseresults suggest that IC is actively transported through theplasma membrane in a form of HCO₃^– probably by some transporterand that the transmembrane Na⁺ gradient is involved in thisIC transport system. Free CO₂ may be hydrated by CA to HCO₃^–and then transported to the cells by this transporter. On the other hand, CO₂ is actively taken up by cells grown withair containing 5% CO₂ (high-CO₂ cells) though the enhancingeffect of Na⁺ was much smaller in high- CO₂ cells than in low-CO₂cells. The initial rate of fixation as a function of internal IC concentrationindicated that the rate of the carboxylation reaction of accumulatedIC is higher in I0W-CO₂ cells than in high-CO₂ cells. The studieswith ethoxyzolamide indicated that even in low-CO₂ cells, CAdoes not function inside Anabaena cells. These results suggestthat inside the low-CO₂ cells of Anabaena, some mediator(s)facilitates the transport of IC to RuBPCase. (Received January 23, 1987; Accepted April 24, 1987)     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Cytoplasmic Alkalization and Cytoplasmic Streaming Induced by Light and Histidine in Leaf Cells of Egeria densa: in vivo 31P-NMR study

Rotational streaming of the cytoplasm including chloroplastswas induced by L-histidine, as well as by light, on the anticlinalface of leaf cells of Egeria densa. In the case of treatmentwith L-histidine some of the chloroplasts remained stationaryon the periclinal face of cells after rotational cytoplasmicstreaming was initiated. However, these chloroplasts were easilydislodged and translocated to the centrifugal end of the histidine-treatedcells by application of a centrifugal force that barely affectedthe location of chloroplasts in cells incubated in the darkwithout L-histidine. This result indicates that the anchoringof chloroplasts was weakened by L-histidine. Thus only the releaseof chloroplasts from anchoring was not enough for initiationof their streaming. The cytoplasmic pH (pH_c) and vacuolar pH(pH_v) were noninvasively monitored by in vivo ³¹P-nuclear magneticresonance (NMR) spectroscopy. Compared with the dark controlvalue, both illumination and treatment with L-histidine increasedthe pH_c by 0.3 units. In contrast, pH_v changed only a littlewith both illumination and treatment with L-histidine. Releaseof chloroplasts from anchoring and initiation of cytoplasmicstreaming are discussed in relation to the increase in pH_c inducedby both light and L-histidine. ⁴ Present address: Department of Cell Biology, National Instituteof Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, 305Japan ⁵ Present address: Marine Biotechnology Institute Co., Ltd.,Head Office, 2-35-10 Hongo, Bunkyo-ku, Tokyo, 113 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Identification and characterization of apolipoprotein(AII-E2-AII) complex in human plasma lipoprotein.

A new apolipoprotein complex designated as the apo(AII-E2-AII) complex was identified in the lipoprotein fractions of human plasma with apoE phenotypes containing apoE2 (E4/E2, E3/E2, and E2/E2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by an immunoblotting assay using anti-apoE or anti-apoAII antibodies, established that the apo(AII-E2-AII) complex, with a molecular weight of 58,000, was identical to the complex consisting of apoE and apoAII, and that it also dissociated following reduction with beta-mercaptoethanol. This new complex was also demonstrated to be distinct from the apo(E-AII) complex and apoE monomer by isoelectric focusing, in the samples that were not treated with beta-mercaptoethanol. In apoE phenotype E3/E2, the apo(AII-E2-AII) complex was primarily included in the high-density lipoprotein (HDL, 1.063 < d < 1.21 g/ml) fraction, but was also observed in a small quantity in the very-low-density lipoprotein (VLDL, d < 1.006 g/ml) fraction. For further characterization, the apo(AII-E2-AII) complex was isolated by preparative SDS-PAGE, and no contamination of apo(E-AII) complex and apoE monomer was detected by immunoblotting assay using an anti-apoE antibody. It was confirmed by an enzyme-linked immunosorbent assay (ELISA) system that a molecular ratio between apoAII monomer and apoE in the isolated apo(AII-E2-AII) complex was approx. 2, when the apo(E-AII) complex was used as a standard with the ratio of 1:1. It indicates that the apo(AII-E2-AII) complex is formed from two molecules of apoAII monomer and one molecule of apoE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii

Carbonic anhydrase purified from the cell surface of Chlamydomonas reinhardtii was inactivated by treatment with dithiothreitol. This treatment caused dissociation of the holoenzyme into 35-kDa (A) and 4-kDa (B) subunits as revealed by SDS/PAGE. The 35-kDa subunit was further separated into two components A1 (35 kDa) and A2 (36.5 kDa) by SDS/PAGE using a gradient gel. These two components have the same amino acid sequence up to at least the 10th amino acid from the N-terminus. The molecular masses were estimated at 76 kDa and 35 kDa for the holoenzyme and the large subunit, respectively, and the molar ratio of the former to the latter at 1:2, by using the techniques of low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC. The molar ratio of the 35-kDa/4-kDa subunits was estimated at 1:1 the gel-filtration HPLC monitored with precision differential refractometry. Atomic-absorption spectrophotometry revealed that the holoenzyme contains two atoms of zinc. These results suggest that the holoenzyme is a heterotetramer composed of two large subunits (A1 and A2) and two small subunits (B).     

Effect of EGF administration on EGF receptor mRNA in hCG producing tumor

In this study we examined the expression of EGF receptor mRNA after EGF administration in hCG producing tumor (choriocarcinoma). We transplanted the tissue of choriocarcinoma into female nude mice and investigated the effects of EGF on the growth of tumors, the binding activity of EGF receptor and the expression of EGF receptor mRNA in the tumor tissues. Two doses of EGF 5.0 micrograms, 50 micrograms and phosphate buffered saline as a control were injected subcutaneously every day for four weeks. Removed tumors were used for immunocytochemical studies and EGF receptor mRNA investigations. HCG and EGF receptors were detected immunocytochemically in the tumor. The low dose EGF employed stimulated the tumor growth while the high dose EGF inhibited the tumor growth compared with that of the control group. The binding activity of EGF receptor and the expression of EGF receptor mRNA also changed in accordance with the stimulation or inhibition of tumor growth. The growth of hCG producing tumor by EGF administration appeared to be dependent upon the binding activity of EGF receptor and the expression of EGF receptor mRNA.     

The peripheral inhibition of the tarsal sugar receptor by sodium chloride in the proboscis extension response of the blowfly,Phormia regina M.

Summary The proboscis extension reponse of the blowfly during stimulation of the tarsal sugar receptors was inhibited by the presence of NaCl. Acceptance thresholds for sucrose in various concentrations of NaCl were measured. The median acceptance thresholds for sucrose in mixtures of 0.01, 0.25, 0.5 and 1.0 M NaCl were 1.8 × 10^–3, 6.0 × 10^–3, 1.2 × 10^–2, and 2.0 × 10^–2 M, respectively. Concentration-response curves for sucrose in the tarsal D-type sugar receptor shifted to the right under the existence of high concentration of NaCl. Number of impulses per D-type sugar receptor at the median acceptance thresholds described above were 7.5, 8.4, 6.8 and 10.4 for the first 0.1 s of stimulation, respectively. The average number was 8.2 impulses per 0.1 s. Comparisons were made between the behavioral acceptance thresholds (1) on one leg exposed to sucrose mixed with 0.01 M NaCl and (2) on two contralateral legs, one of which was exposed to sucrose in 0.01 M NaCl and the other to 0.5 M NaCl alone. The acceptance thresholds from two experiments agreed with each other. The median threshold value was 1.7 × 10^–2 M sucrose. Behavioral inhibition by NaCl in mixtures with sucrose can be explained by its peripheral inhibition of sugar receptors.This research was supported in part by ITO foundation and Scientific Research Fund from the Ministry of Education of Japan.     

Alteration of thyroidal responsiveness to TSH under the influence of circulating thyroid hormone: short feed-back regulatory effect.

In order to examine the hypothesis that the thyroidal responsiveness to TSH is under the influence of thyroid hormone, following the T3 injection to the mice, serum T3 concentrations and the response of thyroid tissue to a fixed dose of TSH in terms of intracellular colloid droplet formation was studied. The colloid droplets induced by TSH was significantly reduced when serum T3 was decreasing, while it was significantly increased when serum T3 was increasing. This results demonstrate for the first time the existence of short feed-back loop regulating intra-thyroidal function by circulating T3. To delineate the possible mechanism of action of T3, the thyroid gland of mouse whose serum T3 concentration was elevated by injecting 50 mug T3, was incubated with TSH in vitro. TSH-induced cyclic AMP generation was not inhibited at all but colloid droplet formation was significantly inhibited in the thyroid tissue of the animal whose serum T3 concentration was enormously high. Thus, it was demonstrated that the site at which T3 affects is beyond cylcic AMP generation but prior to endocytosis, being consistent with our previous results.